Temporal and spatial variation in Anaplasma phagocytophilum infection in Swedish moose (Alces alces).

SUMMARY: The occurrence of Anaplasma phagocytophilum was investigated in spleen and serum samples from Swedish moose (Alces alces) in southern Sweden (island and mainland). Samples were analysed for presence of A. phagocytophilum DNA by real-time PCR (n = 263), and for Anaplasma antibodies with ELISA serology (n = 234). All serum samples had antibodies against A. phagocytophilum. The mean DNA-based prevalence was 26·3%, and significant (P < 0·01) temporal, and spatial variation was found. Island moose had significantly (P < 0·001) higher prevalence of A. phagocytophilum DNA than moose from the mainland areas. Two samples were sequenced to determine genetic variation in the 16S rRNA and groESL genes. Genetic sequence similarity with the human granulocytic anaplasmosis agent, equine granulocytic ehrlichiosis agent, and different wildlife-associated A. phagocytophilum variants were observed in the 16S rRNA and groESL genes. Our study shows that moose are exposed to A. phagocytophilum in Sweden, and represent a potential wildlife reservoir of the pathogen.

Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding.

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gbetagamma-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gbetagamma with high affinity and inhibits the interaction of Gbetagamma with Galpha or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gbetagamma. In this study, we report that Pd was rapidly phosphorylated by Ca(2+)/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gbetagamma binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca(2+)/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivo decreases in Ca(2+) concentration blocked the interaction of Pd with 14-3-3, indicating that Ca(2+) controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca(2+)-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

Applicability of rabbit microsatellite primers for studies of hybridisation between an introduced and a native hare species.

Introduced species may hybridise with relatives in the native fauna or flora and thereby compete for matings and transmit alien DNA. Such interference may contaminate unique genepools, disturb existing ecological balances and may ultimately result in the extinction of the native species. In Sweden, the introduced brown hare (Lepus europaeus Pall.) hybridise with the native mountain hare (L. timidus L.), both relatively common members of the present Swedish fauna. This hybridisation has resulted in the transmission of mitochondrial DNA from the mountain hare to the brown hare, but absence of species differences in karyotype and allozymes have prevented investigations of the amount of nuclear gene flow. More polymorphic genetic markers are needed to analyse evidence of hybridisation in the nuclear genome. The conservation of microsatellite loci across taxa usually enables PCR amplification of microsatellites in closely related species with the same primers. We have used five microsatellite primer pairs, developed for the European wild rabbit (Oryctolagus cuniculus L.) to amplify microsatellites in the two species of hare in Sweden. The obtained allelic variation was used to construct a genetic distance tree based on the amount of shared alleles between all pairs of individuals (shared-allele index). This method offered sufficient differences to arrange all individuals in two groups, one for each species. Identification of individual hybrids based on the number of alleles shared between the species is not possible with these five microsatellite markers.

Mismatch repair is diminished during stationary-phase mutation.

This paper is an invited Response to a recent Commentary [P.L. Foster, Rev. Mut. Res. 436 (1999) 179-184] entitled "Are adaptive mutations due to a decline in mismatch repair? The evidence is lacking". The Commentary argues that no evidence exists supporting the idea that mismatch repair is limiting specifically during stationary-phase mutation. A primary concern of the author is to question the method that we used previously to measure growth-dependent mutation. In this method, mutation rates are calculated using counts of mutant colonies taken at times when those colonies arise, rather than at a predetermined, fixed time. Here we show further data that illustrate why this must be done to ensure accurate mutation measurements. Such accuracy was necessary for our published determination that mismatch repair proteins are not limiting during growth-dependent mutation, but become so during stationary-phase mutation. We review the evidence supporting the idea that stationary-phase reversion of a lac frameshift mutation occurs in an environment of decreased mismatch repair capacity. Those data are substantial. The data presented in the Commentary, in apparent contradiction to this idea, do not justify the conclusion presented there.

Profilaggrin requires both linker and filaggrin peptide sequences to form granules: implications for profilaggrin processing in vivo.

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. Profilaggrin is phosphorylated and contains multiple filaggrin repeats separated by a hydrophobic linker peptide. We have previously shown that filaggrin constructs containing the linker, when transiently transfected into epithelial cells, lead to expression of a protein that resembles keratohyalin (Dale et al. J Invest Dermatol 108:179-187 1997). To characterize further the region(s) of the linker and/or filaggrin that are necessary for granule formation, we generated several mutant constructs from Flag-FG-1, and generated fusions of filaggrin with green fluorescent protein. We also subjected profilaggrin to protein phosphatase 2A treatment and measured its subsequent solubility. We found that granular morphology is not dependent on the linker or conserved phosphorylation sites, nor is solubility affected by protein phosphatase 2A treatment. Granule morphology was abrogated only in a truncated construct, which still contains the linker. A construct consisting of 16 amino acids of filaggrin fused to green fluorescent protein led to rounded and bizarrely shaped transfected cells with compact keratin filaments, suggesting that very little filaggrin sequence is required for keratin filament interaction. Radiolabeled filaggrin-green fluorescent protein constructs specifically bound keratin in overlay assays confirming that the observed cytoskeletal collapse is due to filaggrin-keratin interaction. Our findings indicate that profilaggrin must be extensively processed before it loses both its granule forming ability as well as its insolubility, suggesting that granule formation in vivo correlates with insolubility in vitro. Further, filaggrin retains its ability to bind keratin as it is degraded to smaller peptides.

The immunolocalization and divergent roles of phosducin and phosducin-like protein in the retina.

PURPOSE: These investigations were undertaken to compare and contrast the roles of phosducin and phosducin-like protein in the retina. METHODS: Phosducin and phosducin-like protein were compared in an in vitro assay measuring their inhibition of transducin binding to light-activated rhodopsin. The two proteins were localized within the retina by immunoblot analyses and immunocytochemistry using affinity-purified antibodies with high specificity for each of the two homologs. The sensitivity of phosducin-like protein to phosphorylation was probed using in vitro protein kinase reactions. RESULTS: Phosducin and phosducin-like protein were found to have similar, though not identical, transducin inhibiting activity in vitro. These two proteins were found to be localized dissimilarly within the retina, with spatial overlap limited to the inner segments of the photoreceptors. Phosducin is found exclusively in photoreceptor cells, including the synaptic and nuclear layers, while phosducin-like protein is found throughout the inner retinal layers, most abundantly in the bipolar cells of the inner nuclear layer. Phosducin-like protein is not efficiently phosphorylated by the protein kinases tested, indicating that its regulation differs from that of phosducin. CONCLUSIONS: It appears that phosducin and phosducin-like protein play distinct roles in the retina. While phosducin is likely to be important in feedback regulation of the visual signal, such as in light adaptation, phosducin-like protein probably has little if any function in the phototransduction cascade. Phosducin-like protein may have a role in regulating the processing of visual signals by the neural cells of the inner retina.

Noncontact dipole effects on channel permeation. I. Experiments with (5F-indole)Trp13 gramicidin A channels.

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.

The effects of skin brushing on H reflex amplitude in normal human subjects.

Experiments were performed on twenty-two neurologically normal subjects in order to investigate the effects of skin brushing on H reflex excitability in triceps surae. H reflex amplitude was observed to decrease during brushing of a 2 cm x 10 cm area of skin overlying triceps surae, returning to control levels when brushing ceased. Alterations in the duration of the brushing period did not affect the magnitude of the H reflex inhibition, although increased frequency of brush strokes per minute slightly increased the amount of inhibition observed. Brushing of smaller skin areas overlying the muscle only sometimes resulted in H reflex inhibition. Brushing over other skin areas of the lower limb produced only slight effects on triceps surae H reflex amplitude. Removal of cutaneous input from the skin overlying triceps surae abolished any effects of brushing in this area on H reflex amplitude. These results demonstrate an inhibitory effect of brushing on H reflex excitability in normal subjects. If such results are confirmed in subjects with neurological deficit, they could have implications for the use of brushing in clinical practice.

Transient and heritable mutators in adaptive evolution in the lab and in nature.

Major advances in understanding the molecular mechanism of recombination-dependent stationary-phase mutation in Escherichia coli occurred this past year. These advances are reviewed here, and we also present new evidence that the mutagenic state responsible is transient. We find that most stationary-phase mutants do not possess a heritable stationary-phase mutator phenotype, although a small proportion of heritable mutators was found previously. We outline similarities between this well-studied system and several recent examples of adaptive evolution associated with heritable mutator phenotype in a similarly small proportion of survivors of selection in nature and in the lab. We suggest the following: (1) Transient mutator states may also be a predominant source of adaptive mutations in these latter systems, the heritable mutators being a minority (Rosenberg 1997); (2) heritable mutators may sometimes be a product of, rather than the cause of, hypermutation that gives rise to adaptive mutations.

Mismatch repair protein MutL becomes limiting during stationary-phase mutation.

Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase ("adaptive") mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer.

Genome-wide hypermutation in a subpopulation of stationary-phase cells underlies recombination-dependent adaptive mutation.

Stationary-phase mutation in microbes can produce selected ('adaptive') mutants preferentially. In one system, this occurs via a distinct, recombination-dependent mechanism. Two points of controversy have surrounded these adaptive reversions of an Escherichia coli lac mutation. First, are the mutations directed preferentially to the selected gene in a Lamarckian manner? Second, is the adaptive mutation mechanism specific to the F plasmid replicon carrying lac? We report that lac adaptive mutations are associated with hypermutation in unselected genes, in all replicons in the cell. The associated mutations have a similar sequence spectrum to the adaptive reversions. Thus, the adaptive mutagenesis mechanism is not directed to the lac genes, in a Lamarckian manner, nor to the F' replicon carrying lac. Hypermutation was not found in non-revertants exposed to selection. Therefore, the genome-wide hypermutation underlying adaptive mutation occurs in a differentiated subpopulation. The existence of mutable subpopulations in non-growing cells is important in bacterial evolution and could be relevant to the somatic mutations that give rise to cancers in multicellular organisms.

The occurrence of mountain hare mitochondrial DNA in wild brown hares.

If interspecific hybrids are fertile and backcross to either parental species, transmission of mitochondrial DNA over the species barrier can occur. To investigate if such transmission has occurred between the brown hare Lepus europeus Pall and the mountain hare L. timidus L. in Scandinavia, an analysis of genetic variation in mitochondrial DNA from 36 hares, collected from 15 localities, was performed. Sequence divergence of mtDNA between species was estimated at 8 +/- 1% (SD). Intraspecific mtDNA sequence divergence varied between 0.09 and 0.38% in brown hares and 0.10 and 1.44% in mountain hares. In six out of 18 brown hares examined, two different haplotypes of mountain hare origin were detected, demonstrating a transmission of mtDNA haplotypes from mountain hares to brown hares. The results indicate that interspecific hybridization between the two species occurs in wild populations.

Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.

Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed.

Microheterogeneity of human filaggrin: analysis of a complex peptide mixture using mass spectrometry.

Filaggrin is the product of posttranslational processing of the large, epidermal protein profilaggrin, which consists of 10 or more tandem filaggrin domains plus an amino and a carboxyl domain. According to fragmentary cDNA sequences, the filaggrin domains in the human protein vary at 40% of the amino acid positions; hence, mature filaggrin is a population of homologous but heterogeneous proteins, even within one individual. Available gene sequences give only a limited picture of the heterogeneity of human filaggrin protein because no complete human profilaggrin gene has been sequenced. Questions about the extent of heterogeneity of filaggrin within and between individuals have not been answered, nor have questions concerning the limited proteolytic cleavage of human profilaggrin that generates filaggrin in vivo. In order to address these questions and to provide an analysis of the primary structure of human filaggrins, we employed various methods of mass spectrometry. The intact protein and a tryptic digest of the mixture of human filaggrins were examined by matrix-assisted laser desorption time-of-flight mass spectrometry. Tryptic digests of human filaggrin from single individuals were also separated and analyzed by liquid chromatography/mass spectrometry (LC/MS) (using electrospray mass spectrometry), and specific peptides were identified by tandem mass spectrometry (MS/MS). A robust data analysis program, Sherpa, was developed to facilitate the interpretation of both LC/MS and MS/MS. These experiments show that human filaggrin includes heterogeneity not yet seen in cDNA sequences, but that much structure is highly conserved. Interestingly, we found that the heterogeneity is conserved among individuals. An approximation of the regions linking filaggrins in human profilaggrin is developed. These investigations provide a unique test of the limits of tryptic mapping of complex mixtures using mass spectrometry.

Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.

Human furin catalyzes the proteolytic maturation of many proproteins in the exocytic and endocytic secretory pathways by cleavage at the C-terminal side of the consensus sequence-ArgXaaLys/ArgArg decreases -. Both the trans-Golgi network (TGN) concentration and intracellular routing of furin require sequences in its 56 amino acid cytoplasmic tail. Here, we show that the furin cytoplasmic tail contains multiple trafficking signals. Localization to the TGN requires a cluster of acidic amino acids that, together with a pair of serine residues, forms a casein kinase II (CK II) phosphorylation site. We show that CK II efficiently phosphorylates these serines in vitro, and using a permeabilized cell system we provide evidence that CK II is the in vivo furin kinase. Analysis by mass spectrometry shows that, in vivo, furin exists as di-, mono- and non-phosphorylated forms. Finally, employing (i) furin constructs that mimic either non-phosphorylated or phosphorylated furin and (ii) the phosphatase inhibitor tautomycin, we show that the phosphorylation state of the furin cytoplasmic tail modulates retrieval of the endoprotease to the TGN. Thus, routing of furin is a two-tiered process combining a set of trafficking signals comprised of the primary amino acid sequence of the tail with its phosphorylation state.

Characterization of profilaggrin endoproteinase 1. A regulated cytoplasmic endoproteinase of epidermis.

Profilaggrin, an insoluble precursor of the intermediate filament-associated protein filaggrin, contains multiple internal repeats (PIRs). At terminal differentiation of epidermis, proteolytic processing within a "linker" region of each PIR releases soluble filaggrin in a two-stage process. The first stage endoproteinase (PEP1, profilaggrin endoproteinase 1) cleaves mouse profilaggrin at a subset of the linkers, yielding processing intermediates consisting of several filaggrin repeats. An epidermal endoproteinase that cleaves the requisite linker subset has been purified 4,966-fold from mouse epidermal extracts. SDS-polyacrylamide gel electrophoresis demonstrated a band of molecular mass of 29.5 kDa that correlated with the activity. Labeling with [3H]diisopropylfluorophosphate identified PEP1 as a serine protease; inhibitor studies suggest that it is similar to chymotrypsin, as expected from previous in vivo studies. The purified PEP1 cleaved a peptide derived from profilaggrin (P1) at three residues within and adjacent to a multiple tyrosine sequence, consistent with the in vivo processing sites. No exopeptidase activity was detected. PEP1 is only active toward insoluble profilaggrin, resulting in partial solubilization, consistent with a role in dispersal of profilaggrin during terminal differentiation. In contrast to the specific cleavage of mouse profilaggrin, PEP1 cleaved all linker regions of rat profilaggrin. Studies with phosphorylated P1 suggest that PEP1 specificity may be partly regulated by profilaggrin phosphorylation.

Identification of the amino terminus of human filaggrin using differential LC/MS techniques: implications for profilaggrin processing.

Filaggrin, the intermediate filament aggregating protein of epidermis, is the product of proteolytic processing of the precursor profilaggrin, which consists of 10-20 tandem filaggrin domains. The proteolytic processing sites in mouse and rat profilaggrin have been previously reported. Mouse filaggrin is N-terminally blocked. Rat filaggrin is N-terminally ragged, making it heterogeneous. Human filaggrin, in addition to being N-terminally blocked and potentially ragged at the amino terminus, is heterogeneous due to sequence variation between one filaggrin domain and another along the profilaggrin gene. This complexity has made more difficult the analysis of processing sites in human profilaggrin. We have identified the amino terminus of human filaggrin by applying a general method we have developed for the recognition of amino-terminal peptides in digests of N-terminally blocked proteins. This method compares the peptides in an acetylated and an unacetylated tryptic digest of the protein during their separation by liquid chromatography on-line with electrospray mass spectrometry. In this comparison only the original blocked amino-terminal peptides appear unchanged between the two profiles. Human filaggrin was found to have a heterogeneous N-terminus, as a result both of sequence heterogeneity and of ragged processing; it is blocked by a pyrrolidonecarboxyl group derived from glutamine. By comparison to the termini of rat and mouse filaggrins, implications for the processing of human profilaggrin are discussed.

Crystallization of the MS2 translational repressor alone and complexed to bromouridine.

The coat protein from the MS2 bacteriophage plays a dual role by encapsidating viral RNA and also by binding RNA as a translational repressor. In order to study the isolated dimer in a conformation not influenced by capsid interactions, a mutant molecule was crystallized that is defective in capsid assembly but is an active repressor. The unassembled dimer crystallized in the space group P21212 with a = 76.2, b = 55.7, and c = 28.4 A. In these crystals, monomers were related by twofold symmetry. When this dimer was co-crystallized with 5-bromouridine, crystals formed in space group R3 with a = b = 155.9 A, c = 29.9 A, gamma = 120 degrees; the dimer was the asymmetric unit.